Figure 3.

DJ transcripts, arising from each acrocentric chromosome are essential for nucleolar function. (A) A schematic of the DJ showing location of left arm and right arm transcripts, disnor 187 and disnor 238, respectively. Below, results from RNA FISH performed on hTert-RPE1 cells with BAC clone CH507-145C22 (CU633906) to detect DJ transcripts, labeled in green, together with a human 5′ ETS probe in red. (B) Detection of disnor238 in monochromosomal somatic cell hybrid lines by RT-PCR with primers from exon 1 and 3. A9 and GM09142 (DJ-negative) cells served as negative controls and hTert-RPE1 as a positive control. (C) Twenty-nucleotide chimeric antisense oligonucleotides (ASO1 and 1M) comprise 5-nt 2-O-methoxyribonucleotide segments at both termini and a deoxynucleotide segment containing 10 central nucleotides. Inter-nucleotide linkages were phosphorothioate (*). hTert-RPE1 cells were transfected with ASO1 and 1M and analyzed 24 h later by immunofluorescent staining with antibodies against UBF and NOP52, combined with EU incorporation to monitor ongoing transcription. Effective depletion of DJ transcripts is demonstrated in Supplemental Figure S8. Representative cells, indicated as 1–3, are described in the main text. Quantitation of the experiment is shown below. A further two independent experiments provided essentially the same result (data not shown).